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The zygapophyseal joints of the spine, also known as the facet joints, are paired diarthrodial joints posterior to the intervertebral disc and neural elements. The pathophysiology of facet osteoarthritis (OA), as well as crosstalk between the disc and facets, remains largely understudied compared to disc degeneration. The purpose of this study was to characterize alterations to human facet cartilage and subchondral bone across a spectrum of degeneration and to investigate correlations between disc and facet degeneration. Human lumbar facet articular surfaces from six independent donors were subject to creep indentation mechanical testing to quantify cartilage mechanical properties, followed by microcomputed tomography (µCT) analyses for subchondral bone morphometry. The degenerative state of each articular surface was assessed via macroscopic scoring and via Osteoarthritis Research Society International histopathology scoring. Our data suggest reduced facet cartilage compressive and tensile moduli and increased permeability with increasing degenerative grade, particularly at the lower levels of the spine. µCT analyses revealed spinal level-dependent alterations to the subchondral bone, with an increase in trabecular bone at the L4-L5 level, but a decrease at the upper levels of the lumbar spine with increasing degenerative grade. Cortical bone volume fraction was generally decreased with increasing degenerative grade across spinal levels. Correlation analysis revealed several associations between quantitative measures of disc degeneration and facet OA. This study showed that alterations in the mechanical properties of facet cartilage and in the structural properties of facet subchondral bone correlated with aspects of disc degeneration and were highly dependent on spinal level.

Articular cartilage injury can lead to joint-wide erosion and the early onset of osteoarthritis. To address this, we recently developed a rapid fabrication method to produce patient-specific engineered cartilage tissues to replace an entire articular surface. Here, we extended that work by coupling a mesenchymal stromal cell-laden hydrogel (methacrylated hyaluronic acid) with the porous polycaprolactone (PCL) bone integrating phase and assessed the composition and mechanical performance of these constructs over time. To improve initial construct stability, PCL/hydrogel interface parameters were first optimized by varying PCL pretreatment (with sodium hydroxide before ethanol) before hydrogel infusion. Next, cylindrical osteochondral constructs were formed and cultured in media containing transforming growth factor β3 for up to 8 weeks, with constructs evaluated for viability, histological features, and biochemical content. Mechanical properties were also assessed in axial compression and via an interface shear strength assay. Results showed that the fabrication process was compatible with cell viability, and that construct biochemical content and mechanical properties increased with time. Interestingly, compressive properties peaked at 5 weeks, while interfacial shear properties continued to improve beyond this time point. Finally, these fabrication methods were combined with a custom mold developed from limb-specific computed tomography imaging data to create an anatomic implantable cell-seeded biologic joint surface, which showedmaturation similar to the osteochondral cylinders. Future work will apply these advances in large animal models of critically sized osteochondral defects to study repair and whole joint resurfacing.

OBJECTIVE To elucidate the role of decorin, a small leucine-rich proteoglycan, in the degradation of cartilage matrix during the progression of post-traumatic osteoarthritis (OA). METHODS Three-month-old decorin-null (Dcn-/- ) and inducible decorin-knockout (DcniKO ) mice were subjected to surgical destabilization of the medial meniscus (DMM) to induce post-traumatic OA. The OA phenotype that resulted was evaluated by assessing joint morphology and sulfated glycosaminoglycan (sGAG) staining via histological analysis (n = 6 mice per group), surface collagen fibril nanostructure via scanning electron microscopy (n = 4 mice per group), tissue modulus via atomic force microscopy-nanoindentation (n = 5 or more mice per group) and subchondral bone structure via micro-computed tomography (n = 5 mice per group). Femoral head cartilage explants from wild-type and Dcn-/- mice were stimulated with the inflammatory cytokine interleukin-1β (IL-1β) in vitro (n = 6 mice per group). The resulting chondrocyte response to IL-1β and release of sGAGs were quantified. RESULTS In both Dcn-/- and DcniKO mice, the absence of decorin resulted in accelerated sGAG loss and formation of highly aligned collagen fibrils on the cartilage surface relative to the control (P < 0.05). Also, Dcn-/- mice developed more salient osteophytes, illustrating more severe OA. In cartilage explants treated with IL-1β, loss of decorin did not alter the expression of either anabolic or catabolic genes. However, a greater proportion of sGAGs was released to the media from Dcn-/- mouse explants, in both live and devitalized conditions (P < 0.05). CONCLUSION In post-traumatic OA, decorin delays the loss of fragmented aggrecan and fibrillation of cartilage surface, and thus, plays a protective role in ameliorating cartilage degeneration.

The knee menisci are comprised of two orthogonal collagenous networks – circumferential and radial – that combine to enable efficient load bearing by the tissue in adults. Here, we assessed how the structural and functional characteristics of these networks developed over the course of skeletal maturation and determined the role of these fiber networks in defect tolerance with tissue injury. Imaging of the radial tie fiber (RTF) collagen structure in medial bovine menisci from fetal, juvenile, and adult specimens showed increasing heterogeneity, anisotropy, thickness, and density with skeletal development. Mechanical analysis showed that the tensile modulus in the radial direction did not change with skeletal development, though the resilience (in the radial direction) increased and the tolerance to defects in the circumferential direction decreased, in adult compared to fetal tissues. This loss of defect tolerance correlated with increased order in the RTF network in adult tissue. These data provide new insights into the role of the radial fiber network in meniscus function, will lead to improved clinical decision-making in the presence of a tear, and may improve engineering efforts to reproduce this critical load-bearing structure in the knee.

The meniscus plays a central load bearing role in the knee joint. Unfortunately, meniscus injury is common and can lead to joint degeneration and osteoarthritis. In small animal models, progressive degenerative changes occur with unloading of the meniscus via destabilization of the medial meniscus (DMM). However, few large animal models of DMM exist and the joint-wide initiation of disease has not yet been defined in these models. Thus, the goal of this study was to develop and validate a large-animal model of surgically-induced destabilization of the medial meniscus and to use multi-modal (mechanical, histological, and MRI) and multi-scale (joint to tissue level) quantitative measures to evaluate degeneration in both the meniscus and cartilage. DMM was achieved using an arthroscopic approach in thirteen Yucatan minipigs. One month after DMM, joint contact area decreased and peak pressure increased, indicating altered load transmission as a result of meniscus destabilization. By three months, the joint had adapted to the injury and load transmission patterns were restored to baseline, likely due to the formation and maturation of a fibrovascular scar at the anterior aspect of the meniscus. Despite this, we found a decrease in the indentation modulus of the tibial cartilage and an increase in cartilage histopathology scores at one month compared to Sham operated animals; these deleterious changes persisted through three months. Over this same time course, meniscus remodeling was evident through decreased proteoglycan staining in DMM compared to Sham menisci at both one and three months. These findings support that arthroscopic DMM results in joint degeneration in the Yucatan minipig and provides a new large animal test bed in which to evaluate therapeutics and interventions to treat post-traumatic osteoarthritis (PTOA) that originates from meniscal injury.

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disease characterized by the formation of extra-skeletal bone, or heterotopic ossification (HO), in soft connective tissues such as skeletal muscle. All familial and sporadic cases with a classic clinical presentation of FOP carry a gain-of-function mutation (R206H; c.617G>A) in ACVR1, a cell surface receptor that mediates bone morphogenetic protein (BMP) signaling. The BMP signaling pathway is recognized for its chondro/osteogenic-induction potential, and HO in FOP patients forms ectopic but qualitatively normal endochondral bone tissue through misdirected cell fate decisions by tissue-resident mesenchymal stem cells. In addition to biochemical ligand-receptor signaling, mechanical cues from the physical environment are transduced to activate intracellular signaling, a process known as mechanotransduction, and can influence cell fates. Utilizing an established mesenchymal stem cell model of mouse embryonic fibroblasts (MEFs) from the Acvr1(R206H/+) mouse model that mimics the human disease, we demonstrated that activation of the mechanotransductive effectors Rho/ROCK and YAP1 are increased in Acvr1(R206H/+) cells. We show that on softer substrates, a condition associated with low mechanical signaling, the morphology of Acvr1(R206H/+) cells is similar to the morphology of control Acvr1(+/+) cells on stiffer substrates, a condition that activates mechanotransduction. We further determined that Acvr1(R206H/+) cells are poised for osteogenic differentiation, expressing increased levels of chondro/osteogenic markers as compared to Acvr1(+/+) cells. We also identified increased YAP1 nuclear localization in Acvr1(R206H/+) cells, which can be rescued by either BMP inhibition or Rho antagonism. Our results establish RhoA and YAP1 signaling as modulators of mechanotransduction in FOP and suggest that aberrant mechanical signals, combined with and as a result of the increased BMP pathway signaling through mutant ACVR1, lead to misinterpretation of the cellular microenvironment and a heightened sensitivity to mechanical stimuli that promotes commitment of Acvr1(R206H/+) progenitor cells to chondro/osteogenic lineages.

The incredible mechanical strength and durability of mature fibrous tissues and their extremely limited turnover and regenerative capacity underscores the importance of proper matrix assembly during early postnatal growth. In tissues with composite extracellular matrix (ECM) structures, such as the adult knee meniscus, fibrous (Collagen-I rich), and cartilaginous (Collagen-II, proteoglycan-rich) matrix components are regionally segregated to the outer and inner portions of the tissue, respectively. While this spatial variation in composition is appreciated to be functionally important for resisting complex mechanical loads associated with gait, the establishment of these specialized zones is poorly understood. To address this issue, the following study tracked the growth of the murine meniscus from its embryonic formation through its first month of growth, encompassing the critical time-window during which animals begin to ambulate and weight bear. Using histological analysis, region specific high-throughput qPCR, and Col-1, and Col-2 fluorescent reporter mice, we found that matrix and cellular features defining specific tissue zones were already present at birth, before continuous weight-bearing had occurred. These differences in meniscus zones were further refined with postnatal growth and maturation, resulting in specialization of mature tissue regions. Taken together, this work establishes a detailed timeline of the concurrent spatiotemporal changes that occur at both the cellular and matrix level throughout meniscus maturation. The findings of this study provide a framework for investigating the reciprocal feedback between cells and their evolving microenvironments during assembly of a mechanically robust fibrocartilage tissue, thus providing insight into mechanisms of tissue degeneration and effective regenerative strategies.

Cartilage integration remains a clinical challenge for treatment of focal articular defects. Cartilage exhibits limited healing capacity that declines with tissue maturation. Many approaches have been investigated for their ability to stimulate healing of mature cartilage or integration of repair tissue or tissue-engineered constructs with native cartilage. Growth factors present in immature tissue may enhance chondrogenesis and promote integrative repair of cartilage defects. In this study, we assessed the role of one such factor, fibroblast growth factor 18 (FGF18). Studies using FGF18 have shown a variety of positive effects on cartilage, including stimulation of chondrocyte proliferation, matrix biosynthesis, and suppression of proteinase activity. To explore the role of FGF18 on cartilage defect repair, we hypothesized that treatment with recombinant human FGF18 (sprifermin) would increase matrix synthesis in a defect model, thus improving integration strength. To test this hypothesis, 6 mm cartilage cylinders were harvested from juvenile bovine knees. A central 3 mm defect was created in each explant, and this core was removed and replaced. Resulting constructs were cultured in control or sprifermin-containing medium (weekly 24-h exposure of 100 ng/ml sprifermin) for 4 weeks. Mechanical testing, biochemical analysis, micro-CT, scanning electron microscopy, and histology were used to assess matrix production, adhesive strength, and structural properties of the cartilage-cartilage interface. Results showed greater adhesive strength, increased collagen content, and larger contact areas between core and annular cartilage in the sprifermin-treated group. These findings present a novel treatment for cartilage injuries that have potential to enhance defect healing and lateral cartilage-cartilage integration. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2648-2656, 2018.

Tissue engineering is a promising approach for the repair of articular cartilage defects, with engineered constructs emerging that match native tissue properties. However, the inflammatory environment of the damaged joint might compromise outcomes, and this may be impacted by the choice of cell source in terms of their ability to operate anabolically in an inflamed environment. Here, we compared the response of engineered cartilage derived from native chondrocytes and mesenchymal stem cells (MSCs) to challenge by TNFα and IL-1β in order to determine if either cell type possessed an inherent advantage. Compositional (extracellular matrix) and functional (mechanical) characteristics, as well as the release of catabolic mediators (matrix metalloproteinases [MMPs], nitric oxide [NO]) were assessed to determine cell- and tissue-level changes following exposure to IL-1β or TNF-α. Results demonstrated that MSC-derived constructs were more sensitive to inflammatory mediators than chondrocyte-derived constructs, exhibiting a greater loss of proteoglycans and functional properties at lower cytokine concentrations. While MSCs and chondrocytes both have the capacity to form functional engineered cartilage in vitro, this study suggests that the presence of an inflammatory environment is more likely to impair the in vivo success of MSC-derived cartilage repair. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2901-2910, 2018.

The purpose of study was to investigate the maturation of mesenchymal stem cells (MSC) laden in HA constructs with various combinations of chemically defined medium (CM) components and determine the impact of dexamethasone and serum on construct properties. Constructs were cultured in CM with the addition or withdrawal of media components or were transferred to serum containing media that partially represents an in vivo-like condition where pro-inflammatory signals are present. Constructs cultured in CM+ (CM with TGF-β3) and DEX- (CM+ without dexamethasone) conditions produced robust matrix, while those in ITS/BSA/LA- (CM+ without ITS/BSA/LA) and Serum+ (10% FBS with TGF-β3) produced little matrix. While construct properties in DEX- were greater than those in CM+ at 4 weeks, properties in CM+ and DEX- reversed by 8 weeks. While construct properties in DEX- were greater than those in CM+ at 4 weeks, the continued absence or removal of dexamethasone resulted in marked GAG loss by 8 weeks. Conversely, the continued presence or new addition of dexamethasone at 4 weeks further improved or maintained construct properties through 8 weeks. Finally, when constructs were converted to Serum (in the continued presence of TGF-β3 with or without dexamethasone) after pre-culture in CM+ for 4 weeks, GAG loss was attenuated with addition of dexamethasone. Interestingly, however, collagen content and type was not impacted. In conclusion, dexamethasone influences the functional maturation of MSC-laden HA constructs, and may help to maintain properties during long-term culture or with in vivo translation by repressing pro-inflammatory signals. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1717-1727, 2018.

Genetic tools such as the Cre‐Lox reporter system are powerful aids for tissue‐specific cell tracking. For example, it would be useful in examining intervertebral disc (IVD) cell populations in normal and diseased states. A Cre recombinase and its recognition site, loxP have been adapted from the bacteriophage for use in genetic manipulation. The reporter mice used here express the red fluorescent protein, tdTomato with flanking LoxP sites (Rosa26 TdTomato mice). We compared two different Collagen type II (Col2) promoter constructs that drive Cre‐recombinase expression in mice: (a) Col2‐Cre, which allows constitutive Cre‐recombinase expression under the control of the Col2 promoter/enhancer and (b) Col2‐CreER, which contains a shorter promoter/enhancer region than Col2‐Cre, but has human estrogen binding elements that bind tamoxifen, resulting in Cre‐recombinase expression. The goal of the study is to characterize Cre‐recombinase distribution pattern in Col2‐Cre and Col2‐CreER mice using tdTomato as reporter in the spine. The expression patterns of these two mice were further compared with Col2 gene expression in the native mouse NP and AF tissues by real‐time PCR. We crossed Col2‐Cre mice or Col2‐CreER mice with the tdTomato reporter mice, and compared the tdTomato expression patterns. Col2‐CreER/tdTomato mice were injected with tamoxifen at postnatal day 7 to activate the Cre‐recombinase. TdTomato in the constitutively active Col2‐Cre mice was detected in the nucleus pulposus (NP), the entire annulus fibrosus (AF), and in cartilaginous endplate and growth plate cells in the lower lumbar and coccygeal spine. In contrast, when Col2‐CreER activity was induced by tamoxifen at P7, tdTomato was limited to the inner AF, and was absent from the NP. We have described the differences in Col2 reporter gene expression, in Col2‐Cre/tdTomato and Col2‐Cre‐ER/tdTomato mouse IVD. The information provided here will help to guide future investigations of IVD biology.

Cartilage injuries and subsequent tissue deterioration impact millions of patients. Since the regeneration of functional hyaline cartilage remains elusive, methods to stabilize the remaining tissue, and prevent further deterioration, would be of significant clinical utility and prolong joint function. Finite element modeling shows that fortification of the degenerate cartilage (Reinforcement) and reestablishment of a superficial zone (Sealing) are both required to restore fluid pressurization within the tissue and restrict fluid flow and matrix loss from the defect surface. Here, a hyaluronic acid (HA) hydrogel system is designed to both interdigitate with and promote the sealing of the degenerated cartilage. Interdigitating fortification restores both bulk and local pericellular tissue mechanics, reestablishing the homeostatic mechanotransduction of endogenous chondrocytes within the tissue. This HA therapy is further functionalized to present chemo mechanical cues that improve the attachment and direct the response of mesenchymal stem/stromal cells at the defect site, guiding localized extracellular matrix deposition to "seal" the defect. Together, these results support the therapeutic potential, across cell and tissue length scales, of an innovative hydrogel therapy for the treatment of damaged cartilage.

Rotator cuff tendon tears are one of the most common shoulder pathologies, especially in the aging population. Due to a poor healing response and degenerative changes associated with aging, rotator cuff repair failure remains common. Although cell-based therapies to augment rotator cuff repair appear promising, it is unknown whether the success of such a therapy is age-dependent. We hypothesized that autologous cell therapy would improve tendon-to-bone healing across age groups, with autologous juvenile cells realizing the greatest benefit. In this study, juvenile, adult, and aged rats underwent bilateral supraspinatus tendon repair with augmentation of one shoulder with autologous tendon-derived cell-seeded polycaprolactone scaffolds. At 8 weeks, shoulders treated with cells in both juvenile and aged animals exhibited increased cellularity, increased collagen organization, and improved mechanical properties. No changes between treated and control limbs were seen in adult rats. These findings suggest that cell delivery during supraspinatus repair initiates earlier matrix remodeling in juvenile and aged animals. This may be due to the relative "equilibrium" of adult tendon tissue with regards to catabolic and anabolic processes, contrasted with actively growing juvenile tendons and degenerative aged tendons. This study demonstrates the potential for autologous cell-seeded scaffolds to improve repairs in both the juvenile and aged population. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1250-1257, 2017.

Improved diagnostic measures for intervertebral disc degeneration are necessary to facilitate early detection and treatment. The aim of this study was to correlate changes in mechanical and biochemical properties with the quantitative MRI parameters T2 and T1ρ in rabbit lumbar discs using an ex vivo chymopapain digestion model. Rabbit lumbar spinal motion segments from animals less than 6 months of age were injected with 100 μl of saline (control) or chymopapain at 3, 15, or 100 U/ml (n = 5 per group). T2 and T1ρ MRI series were obtained at 4.7T. Specimens were mechanically tested in tension-compression and creep. Normalized nucleus pulposus (NP) water and GAG contents were quantified. Stepwise multiple linear regression was performed to determine which parameters contributed significantly to changes in NP T2 and T1ρ. When all groups were included, multiple regression yielded a model with GAG, compressive modulus, and the creep time constants as variables significantly impacting T2 (multiple r(2) = 0.64, p = 0.006). GAG and neutral zone (NZ) modulus were identified as variables contributing to T1ρ (multiple r(2) = 0.28, p = 0.08). When specimens with advanced degeneration were excluded from the multiple regression analysis, T2 was significantly predicted by compressive modulus, τ1, and water content (multiple r(2) = 0.71, p = 0.009), while no variables were significant predictors in the model for T1ρ. These results indicate that quantitative MRI can detect changes in the mechanical and biochemical properties of the degenerated disc. T2 may be more sensitive to early stage degenerative changes than T1ρ, while both quantitative MRI parameters are sensitive to advanced degeneration. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1382-1388, 2016.

The ability to detect meniscus defects by magnetic resonance arthrography (MRA) can be highly variable. To improve the delineation of fine tears, we synthesized a cationic gadolinium complex, (Gd-DOTA-AM4 )(2+) , that can electrostatically interact with Glycosaminoglycans (GAGs). The complex has a longitudinal relaxivity (r1) of 4.2 mM(-1) s(-1) and is highly stable in serum. Its efficacy in highlighting soft tissue tears was evaluated in comparison to a clinically employed contrast agent (Magnevist) using explants obtained from adult bovine menisci. In all cases, Gd-DOTA-AM4 appeared to improve the ability to detect the soft tissue defect by providing increased signal intensity along the length of the tear. Magnevist shows a strong signal near the liquid-meniscus interface, but much less contrast is observed within the defect at greater depths. This provides initial evidence that cationic contrast agents can be used to improve the diagnostic accuracy of MRA. Copyright © 2016 John Wiley & Sons, Ltd.

Cells respond to physical cues in their microenvironment. These cues result in changes in cell behavior, some of which are transient, and others of which are permanent. Understanding and leveraging permanent alteration of cell behavior induced by mechanical cues, or "mechanical memories," is an important aim in cell and tissue engineering. Herein, this paper reviews the existing literature outlining how cells may store memories of biophysical cues with a specific focus on the nucleus, the storehouse of information in eukaryotic cells. In particular, this review details mechanically driven adaptations in nuclear structure and genome organization and outlines potential mechanisms by which mechanical memories may be encoded within the structure and organization of the nucleus and chromatin.

Objectives/Hypothesis To develop an effective rabbit model of in vitro- and in vivo-derived tissue-engineered cartilage for laryngotracheal reconstruction (LTR). Study Design 1) Determination of the optimal scaffold 1% hyaluronic acid (HA), 2% HA, and polyglycolic acid (PGA) and in vitro culture time course using a pilot study of 4 by 4-mm in vitro-derived constructs analyzed on a static culture versus zero-gravity bioreactor for 4, 8, and 12 weeks, with determination of compressive modulus and histology as outcome measures. 2) Three-stage survival rabbit experiment utilizing autologous auricular chondrocytes seeded in scaffolds, either 1% HA or PGA. The constructs were cultured for the determined in vitro time period and then cultured in vivo for 12 weeks. Fifteen LTRs were performed using HA cartilage constructs, and one was performed with a PGA construct. All remaining specimens and the final reconstructed larynx underwent mechanical testing, histology, and glycosaminoglycan (GAG) content determination, and then were compared to cricoid control specimens (n = 13) and control LTR using autologous thyroid cartilage (n = 18). Methods 1) One rabbit underwent an auricular punch biopsy, and its chondrocytes were isolated and expanded and then encapsulated in eight 4 by 4-mm discs of 1% HA, 2% HA, PGA either in rotary bioreactor or static culture for 4, 8, and 12 weeks, respectively, with determination of compressive modulus, GAG content, and histology. 2) Sixteen rabbits underwent ear punch biopsy; chondrocytes were isolated and expanded. The cells were seeded in 13 by 5 by 2.25-mm UV photopolymerized 1% HA (w/w) or calcium alginate encapsulated synthetic PGA (13 × 5 × 2 mm); the constructs were then incubated in vitro for 12 weeks (the optimal time period determined above in paragraph 1) on a shaker. One HA and one PGA construct from each animal was tested mechanically and histologically, and the remaining eight (4 HA and 4 PGA) were implanted in the neck. After 12 weeks in vivo, the most optimal-appearing HA construct was used as a graft for LTR in 15 rabbits and PGA in one rabbit. The seven remaining specimens underwent hematoxylin and eosin, Safranin O, GAG content determination, and flexural modulus testing. At 12 weeks postoperative, the animals were euthanized and underwent endoscopy. The larynges underwent mechanical and histological testing. All animals that died underwent postmortem examination, including gross and microhistological analysis of the reconstructed airway. Results Thirteen of the 15 rabbits that underwent LTR with HA in vitro- and in vivo-derived tissue-engineered cartilage constructs survived. The 1% HA specimens had the highest modulus and GAG after 12 weeks in vitro. The HA constructs became well integrated in the airway, supported respiration for the 12 weeks, and were histologically and mechanically similar to autologous cartilage. Conclusions The engineering of in vitro- and in vivo-derived cartilage with HA is a novel approach for laryngotracheal reconstruction. The data suggests that the in vitro- and in vivo-derived tissue-engineered approaches may offer a promising alternative to current strategies used in pediatric airway reconstruction, as well as other head and neck applications. Level of Evidence NA. Laryngoscope, 2015

Supraspinatus tears often result in the setting of chronic tendinopathy. However, the typical repair model utilizes an acute injury. In recognition of that distinction, our laboratory developed an overuse animal model; however it is unclear whether induced overuse is necessary in the repair model. We studied the repair properties of overuse-induced tendons compared to normal tendons. We hypothesized that histological and mechanical properties would not be altered between the overuse-induced and normal tendons 1 and 4 weeks after repair. Thirty-one adult male Sprague-Dawley rats were subjected to either overuse or cage activity for 4 weeks prior to bilateral supraspinatus tendon repair surgery. Rats were sacrificed at 1 and 4 weeks post-surgery and evaluated for histology and mechanics. Results at 1 week showed no clear histologic changes, but increased inflammatory protein expression in overuse tendons. At 4 weeks, percent relaxation was slightly increased in the overuse group. No other alterations in mechanics or histology were observed. Our results suggest that the effects of the surgical injury overshadow the changes evoked by overuse. Because clinically relevant mechanical parameters were not altered in the overuse group, we conclude that when examining tendons 4 weeks after repair in the classic rat supraspinatus model, inducing overuse prior to surgery is likely to be unnecessary.

Degeneration of the intervertebral discs is strongly implicated as a cause of low back pain. Since current treatments for discogenic low back pain show poor long-term efficacy, a number of new biological strategies are being pursued. For such therapies to succeed, it is critical that they be validated in conditions that mimic the unique biochemical microenvironment of the nucleus pulposus (NP), which include low oxygen tension. Therefore, the objective of this study was to investigate the effects of oxygen tension on NP cell functional extracellular matrix elaboration in 3D culture. Bovine NP cells were encapsulated in agarose constructs and cultured for 14 or 42 days in either 20% or 2% oxygen in defined media containing transforming growth factor beta-3. At each time point, extracellular matrix composition, biomechanics, and mRNA expression of key phenotypic markers were evaluated. Results showed that while bulk mechanics and composition were largely independent of oxygen level, low oxygen promoted improved restoration of the NP phenotype, higher mRNA expression of extracellular matrix and NP specific markers, and more uniform matrix elaboration. These findings indicate that culture under physiological oxygen levels is an important consideration for successful development of cell and growth factor-based regenerative strategies for the disc.

Mesenchymal stem cells (MSCs) hold great promise for regenerative therapies and tissue engineering applications given their multipotential differentiation capacity. However, MSC isolation and expansion are typically performed on super-physiologically stiff tissue culture plastic (TCP), which may alter their behavior and lead to unintended consequences upon implantation. In contrast, electrospun nanofibrous scaffolds possess physical and mechanical properties that are similar to that of native tissue. In this study, we investigated whether isolation and expansion of juvenile bovine MSCs directly onto electrospun nanofibrous scaffolds better preserves MSC phenotype and stemness compared to TCP. Our data show that culture of MSCs on electrospun scaffolds reduces proliferation, decreases cellular senescence, and better maintains stemness compared to cells isolated and expanded on TCP, likely due to a reduction in cell contractility. Furthermore, in contrast to electrospun scaffolds, TCP biased MSCs towards a fibrotic phenotype that persisted even after the cells were reseeded onto a different substrate. Cells pre-cultured on electrospun scaffolds exhibited a heightened response to mechanical stimuli and greater chondrogenesis in methacrylated hyaluronic acid hydrogels. These data suggest that alternative substrates that better approximate the native cell environment should be used to preserve endogenous MSC behavior and may improve their success in tissue engineering applications. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:808-815, 2018.

Magnetic resonance imaging (MRI) with T2-weighting is routinely performed to assess intervertebral disc degeneration. Standard clinical evaluations of MR images are qualitative, however, and do not focus on region-specific alterations in the disc. Utilizing a rabbit needle puncture model, T2 mapping was performed on injured discs to develop a quantitative description of the degenerative process following puncture. To do so, an 18G needle was inserted into four discs per rabbit (L3/L4 to L6/L7) and T2 maps were generated pre- and 4 weeks post-injury. Individual T2 maps were normalized to a disc-specific coordinate system and then averaged for pre- and post-injury population composite T2 maps. We also developed a method to automatically segment the nucleus pulposus by fitting the NP region of the T2 maps with modified 2-D and 3-D Gaussian distribution functions. Puncture injury produced alterations in MR signal intensity in a region-specific manner mirroring human degeneration. Population average T2 maps provided a quantitative representation of the injury response, and identified deviations of individual degenerate discs from the pre-injury population. We found that the response to standardized injury was modest at lower lumbar levels, likely as a result of increased disc dimensions. These tools will be valuable for the quantitative characterization of disc degeneration in future clinical and pre-clinical studies. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:140–148, 2015.

Fibrous scaffolds fabricated via electrospinning are being explored to repair injuries within dense connective tissues. However, there is still much to be understood regarding the appropriate scaffold properties that best support tissue repair. In this study, the influence of the stiffness of electrospun fibers on cell invasion into fibrous scaffolds is investigated. Specifically, soft and stiff electrospun fibrous networks are fabricated from crosslinked methacrylated hyaluronic acid (MeHA), where the stiffness is altered via the extent of MeHA crosslinking. Meniscal fibrochondrocyte (MFC) adhesion and migration into fibrous networks is investigated, where the softer MeHA fibrous networks are easily deformed and densified through cellular tractions and the stiffer MeHA fibrous networks support ~50% greater MFC invasion over weeks when placed adjacent to meniscal tissue. When the scaffolds are sandwiched between meniscal tissues and implanted subcutaneously, the stiffer MeHA fibrous networks again support enhanced cellular invasion and greater collagen deposition after 4 weeks when compared to the softer MeHA fibrous networks. These results indicate that the mechanics and deformability of fibrous networks likely alter cellular interactions and invasion, providing an important design parameter towards the engineering of scaffolds for tissue repair.

The annulus fibrosus (AF) of the intervertebral disk plays a critical role in vertebral load transmission that is heavily dependent on the microscale structure and composition of the tissue. With degeneration, both structure and composition are compromised, resulting in a loss of AF mechanical function. Numerous tissue engineering strategies have addressed the issue of AF degeneration, but few have focused on recapitulation of AF microstructure and function. One approach that allows for generation of engineered AF with appropriate (+/-)30° lamellar microstructure is the use of aligned electrospun scaffolds seeded with mesenchymal stem cells (MSCs) and assembled into angle-ply laminates (APL). Previous work indicates that opposing lamellar orientation is necessary for development of near native uniaxial tensile properties. However, most native AF tensile loads are applied biaxially, as the disk is subjected to multi-axial loads and is constrained by its attachments to the vertebral bodies. Thus, the objective of this study was to evaluate the biaxial mechanical response of engineered AF bilayers, and to determine the importance of opposing lamellar structure under this loading regime. Opposing bilayers, which replicate native AF structure, showed a significantly higher modulus in both testing directions compared to parallel bilayers, and reached ∼60% of native AF biaxial properties. Associated with this increase in biaxial properties, significantly less shear, and significantly higher stretch in the fiber direction, was observed. These results provide additional insight into native tissue structure-function relationships, as well as new benchmarks for engineering functional AF tissue constructs.

The use of high-throughput screening (HTS) techniques has long been employed by the pharmaceutical industry to increase discovery rates for new drugs that could be useful for disease treatment, yet this technology has only been minimally applied in other applications such as in tissue regeneration. In this work, an assay for the osteogenic differentiation of human mesenchymal stem cells (hMSCs) was developed and used to screen a library of small molecules for their potential as either promoters or inhibitors of osteogenesis, based on levels of alkaline phosphatase activity and cellular viability. From a library of 1,040 molecules, 36 promoters, and 20 inhibitors were identified as hits based on statistical criteria. Osteopromoters from this library were further investigated using standard culture techniques and a wider range of outcomes to verify that these compounds drive cellular differentiation. Several hits led to some improvement in the expression of alkaline phosphatase, osteogenic gene expression, and matrix mineralization by hMSCs when compared to the standard dexamethasone supplemented media and one molecule was investigated in combination with a recently identified biodegradable and osteoconductive polymer. This work illustrates the ability of HTS to more rapidly identify potential molecules to control stem cell differentiation.

The knee menisci are wedge-shaped semilunar fibrocartilaginous structures that reside between the femur and tibia and function to transmit and distribute load. These structures have characteristics of both fibrous and cartilaginous tissues. The cartilage-like inner region and the fibrous vascularized outer region each has a distinct extracellular matrix, and resident meniscal fibrochondrocytes (MFCs) with distinct morphologies dependent on their location. Damage to the meniscus is common, and disruption of tissue structure and function result in erosion of the underlying articular cartilage. It has been observed that damage in the vascular periphery undergoes spontaneous repair, whereas damage of the inner region does not heal. While vascularity of the peripheral region plays a role in healing, recent findings have also suggested that local cellular composition influences local healing capacity. This study examined the variation in multipotential characteristics of cell populations isolated from different regions of the bovine meniscus. MFCs were isolated from the outer (vascular), inner (avascular), and horn (mixed) regions and induced toward chondrogenic, adipogenic, and osteogenic lineages. The results of this study suggest that MFCs from all regions of the meniscus possess a multilineage differentiation capability, particularly toward chondrogenesis and adipogenesis. MFCs from the outer region were most plastic, differentiating along all three mesenchymal lineages. These findings may underlie the experimental observation of improved integration of meniscus grafts from the outer zone and may have implications for developing strategies of cell-based meniscus repair.

Inspired by the depth-dependent inhomogeneity of articular cartilage, it was hypothesized that a novel layered agarose technique, using a 2% (wt/vol) top and a 3% (wt/vol) bottom layer, would create an inhomogenous tissue construct with distinct material properties in conjoined regions. The biochemical and mechanical development of these constructs was observed alongside uniform 2% and 3% constructs. Initially, uniform 3% agarose disks had the highest bulk Young’s modulus (EY � 28 kPa) of all groups. After 28 days of culture in 20% FBS-containing media, however, uniform 2% chondrocyte-seeded constructs achieved the highest Young’s modulus compared to bilayered and 3% agarose disks. Though all three groups contained similar GAG content (� 1.5% ww), uniform 2% agarose disks on day 28 possessed the highest collagen content (� 1% ww). Unlike in either homogeneous construct type, microscopic analysis of axial strain fields in bilayered constructs in response to applied static compression revealed two mechanically disparate regions on day 0: a softer 2% layer and a stiffer 3% layer. With time in culture, this inhomogeneity became less distinct, as indicated by increased continuity in both the local displacement field and local EY, and depended on the level of FBS supplementation of the feed media, with lower FBS concentrations (10%) more closely maintaining the original distinction of material properties. These results shed positive light on a layered agarose technique for the production of inhomogeneous bilayered chondrocyte-seeded agarose constructs with applications for investigations of chondrocyte mechanotransduction and for possible use in the tissue engineering of inhomogeneous articular cartilage constructs. � 2004 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved.